Firefly luciferase exhibits burst kinetics: upon contact with the substrate, an initial burst of light is followed by a significant drop in the rate of light output.
There has been great disagreement as to the molecular basis for the observation that luciferase light output decreases (fairly rapidly) over time, giving rise to “burst kinetics”. Three explanations have been proposed: 1) product inhibition by oxyluciferin, 2) inhibition by the off-pathway formation of dehydroluciferyl-AMP, and 3) free-radical damage of the luciferase by the light-generating reaction itself. These explanations are not mutually exclusive.
Oxyluciferin itself has an affinity for luciferase that is similar to D-luciferin, while the binding affinity of AMP is significantly lower. For these products to have a substantial effect on the light output of luciferase by virtue of product inhibition, a cooperative effect on dissociation, or the trapping of the luciferase in an inactive oxyluciferin-ATP-bound state are the most likely possible scenarios.
Another potential source of product inhibition is from the “dark” side product, L-AMP. This compound can be produced from LH2-AMP by oxidation to give the dehydroluciferin rather than formation of the excited state oxyluciferin. No light is produced, and the phosphate ester linkage is not cleaved. This “bidentate” product therefore has significantly higher binding affinity than D-luciferin, oxyluciferin, or AMP alone.
When firefly luciferase and D-luciferin are treated with coenzyme A (CoASH), some rescue of light output is obtained. This has been interpreted as arising from the conversion of L-AMP into L-CoA and AMP, which have lower binding affinity and can therefore more readily diffuse away from the binding pocket.
Light emission from firefly luciferase is fundamentally limited by its access to D-luciferin and the inherent photophysical properties of the D-luciferin substrate (Reddy et al., J. Am. Chem. Soc 132, 13586-13587, 2010). Replacement of the 6′-hydroxyl group of D-luciferin with a 6′-amino group results in red-shifted light emission (White et al., J. Am. Chem. Soc. 88, 2015-2019, 1966) and higher affinity for luciferase, but lower maximal light emission and lower cell-permeability (Shinde et al., Biochemistry 45, 11103-12, 2006). Although D-luciferin is the superior substrate for maximal light emission under most conditions, the unique chemistry of 6′-aminoluciferin has expanded the scope of luciferase applications. For example, the liberation of 6′-aminoluciferin from “dark” pro-luciferin protease substrates has been exploited to allow the coupled bioluminescent detection of protease activity, both in vitro (Monsees et al., Anal. Biochem. 221, 329-34, 1994; Moravec et al., Anal. Biochem 387, 294-302, 2009) and in vivo (Shah et al., Mol Ther 11, 926-931, 2005; Dragulescu-Andrasi et al., Bioconjug Chem 20, 1660-1666, 2009; Hickson et al., Cell Death Differ 17, 1003-1010, 2010; Scabini et al., Apoptosis 16, 198-207, 2011).
6′-alkylaminoluciferins can also be substrates for luciferase (Woodroofe et al., Biochemistry 47, 10383-1039, 2008; Reddy et al., J. Am. Chem. Soc 132, 13586-13587, 2010; Takakura et al., Chem Asian J 5, 2053-2061, 2010). These substrates generally have even higher affinity for luciferase than 6′-aminoluciferin, and emit light at even longer wavelengths. Many modifications are tolerated, including long-chain 6′-alkylaminoluciferins, 5′,6′-cyclic alkylaminoluciferins, and even dialkylaminoluciferins. Synthetic modulation of the properties of these molecules thus presents an opportunity to develop new bioluminescent probes and to optimize luciferase light output for different applications. However, with wild-type Photinus pyralis firefly luciferase, most of these substrates give a rapid burst of light followed by weak sustained emission (Reddy et al., J. Am. Chem. Soc 132, 13586-13587, 2010).
The detergent-stable proprietary mutant luciferase Ultra-Glo (Promega) is capable of high sustained light emission with aminoluciferin substrates, particularly in combination with the P450-Glo buffer (Woodroofe et al., Biochemistry 47, 10383-1039, 2008; Reddy et al., J. Am. Chem. Soc 132, 13586-13587, 2010). The use of aminoluciferins with this luciferase and buffer therefore has potential for novel in vitro screening applications, such as the coupled detection of enzymatic activity (Fan and Wood, Assay Drug. Dev. Technol. 5, 127-136, 2007). However, Ultra-Glo is a proprietary luciferase reagent that is not available as a genetic construct that can be expressed in cells. Furthermore, the detergent stability of Ultra-Glo and the use of the P450-Glo buffer are important for the light emission behavior. Cellular and in vivo applications such as the detection of gene expression (de Wet et al., Mol Cell Biol 7, 725-737, 1987) and bioluminescent imaging (Prescher and Contag, Curr. Op. Chem. Biol. 14, 80-89, 2010) necessitate a genetically-encodable luciferase that is capable of efficient utilization of aminoluciferins under physiological buffer conditions.
To monitor gene expression in mammalian cells or perform bioluminescence imaging in whole organisms, a genetically-encoded luciferase capable of efficient light emission with aminoluciferins is necessary.